The present invention relates generally to fluorescence detection systems, and more particularly to reducing or eliminating cross-talk and extending the dynamic range of such systems.
Optical fluorescence detection technologies are becoming more and more key tools for advancing bioscience discoveries. And, as the pace of scientific research increases, the demand for sensitivity, quantification, speed, and automation of such tools increases. One detection system, the LI-COR Odyssey® scanner (see, e.g., U.S. Pat. No. 6,495,812), uses a sensitive optical detection system, optimized for the Near Infra-Red spectral region, where it is well known that most biological samples and most materials have low auto-fluorescence, and delivers the sensitivity needed for high demanding applications. The system has nearly 15-bit (>4 orders of magnitude) of dynamic range within the same scan. This is more than sufficient for most applications. However, the levels of fluorescence signals can vary significantly from application to application and therefore there is currently a need to select a gain setting that allows for capturing most of the dynamic range without much saturation. FIG. 1a shows an image of a 2× dilution with saturated spots. The rows of region “A” are of the same 800-channel dilution. FIG. 1b shows a profile that runs across the dilution (i.e. both rows) and indicates that 4 spots are totally saturated and one spot partially saturated.
The region labeled “B” shows some channel cross-talk from the 700-channel that results in saturating the 800-detector. At the shown saturated spot in region “B”, there exists another dilution, but with a 700-dye label (IRDye® 700DX, which is excitable only very minimally by the 780 nm laser). But, the saturation of the 800 detector at those locations is an indication of a relatively strong fluorescence signal resulting from the 700 excitation (i.e. 680 nm laser). A similar channel cross-talk occurs from the 800 excitation into the 700-channel detection. Reducing such a cross-talk enhances the sensitivity of both channels across the whole field.
Odyssey® uses pre-set excitation laser power and Avalanche Photo-Diodes (APDs) as detectors for detecting emitted fluorescence. The signal level provided by the system depends on the laser power, the fluorescence concentration, detection collection efficiency, and detector gain settings. The former three are set by design and the gain setting is adjustable. This system-level gain includes an amplification gain of the APD itself followed by an electronic amplification stage. Currently, the range of gain settings available takes advantage of both components, and allows for more than 1000× system-level gain change. It is desirable, however, to maintain the APD gain level at a single setting so that its sensitivity performance is maintained over the wider dynamic range. Fixing the APD gain leaves only the electronic amplification to adjust, which results in a limited extension of the dynamic range. Furthermore, relatively strong fluorescence signals can saturate the APD and in that case, the electronic gain can not result in any useful extension of the dynamic range. There is therefore a need to extend the dynamic range capability of a scanner, such as Odyssey®, without changing the sensitivity of the detector.
The Odyssey® scanning design benefits in a number of ways from exciting the same location of a sample with two laser colors. The optical filtering and the modulation-demodulation technique allows for efficient separation of emitted fluorescence stimulated by both lasers giving two images simultaneously: 700 and 800 channels. However, when the amount of emitted fluorescence is high, residual fluorescence leakage from one channel into another can result in limiting the dynamic range of the APD. For example, the 800-channel APD can receive leakage from 700-channel fluorescence than can saturate it even if there is no 800-channel fluorescence, and demodulating with the 800 laser modulation frequency does not help. This case is clearly shown in the spots in area “B” in FIG. 1a. The image is an 800-channel image and the shown spots contain 700-dye only, but with relatively high concentrations. It clearly shows the 800-channel detector is saturated even though there is no 800 fluorescence. This cross-channel effect can be reduced by changing the APD gain described above, but this affects its sensitivity to 800-fluorescence, the desired fluorescence in that channel. It is, therefore, desirable to minimize or eliminate this cross-channel effect while maintaining the APD gain setting at its, high, sensitive setting. It is further desirable to accomplish this together with extending the dynamic range of the system.
U.S. Pat. No. 7,463,357 extends the dynamic range of a chemical array reader by splitting the detection light into two light beams and detecting them with different types of detectors having overlapping dynamic ranges. This does not reduce the channel cross-talk described above and requires both detectors to have similar optimum operating gain settings. Furthermore, splitting the light into two beams effectively results in a reduction of the otherwise obtainable sensitivity. It is desirable to maintain or enhance the sensitivity, not reduce it. Corson (U.S. Pat. Nos. 6,952,008 and 6,806,460) also teaches a fluorescence detection technique based on the same idea of splitting the collected fluorescence light within a spectral range (color) into two portions and detecting them with detectors of different dynamic ranges and combing both measurements to produce an image with higher dynamic range.
U.S. Pat. No. 7,054,003 teaches how to read different regions of a chemical array with light of different intensities and simultaneously detecting light emitted from the different regions, as a way to image an array with a wide dynamic range. This does not solve the limitation described above, which desires to maintain the illumination of and detection from the same location. Applying the different intensity idea at the same location and detecting them both at the same time does not work with the same excitation and emission wavelength ranges and results in more of the channel cross-talk problem. Even if combined with other techniques, such as described by U.S. patent application Ser. No. 11/036,571, to split the beam into portions and detect them separately, requires adding other detectors and associated optics for each color, a costly endeavor.
U.S. Pat. No. 7,361,472 provides a method for extending the dynamic range of scattered light measurement based on measuring the same light multiple times using different filters to reduce signal levels and then combines the measurements using pre-determined filter optical density ratios. In addition to the fact that imaging in fluorescence presents different challenges that the invention does not address, the idea of re-measuring the same light by using filters to block part of the light being detected does not solve the channel cross-talk problem.
U.S. Pat. No. 6,870,166 and application Ser. No. 11/344,773 describe methods of extending the dynamic range of reading chemical array by scanning the array twice at different gain settings. Although, this avoids having to split the collected light into portion(s), it still does not work if detectors, such as APDs, are to be operated at the same optimized gain setting, as described above. Furthermore, this method does not change any channel cross-talk caused by simultaneous multi-color detection, such as desired here.
U.S. Pat. No. 6,320,196 presents a solution to dye cross-talk that can work for channel cross-talk, but it is based on spatially separating different color optics, i.e. focusing lasers at separate spots and collecting emitted fluorescence by separate optics and detectors. This does not have the advantage of compact, cost-effective of single-spot scanning and techniques that work for separated optics do not work for combined optics.
Therefore it is desirable to provide systems and methods that overcome the above and other problems. In particular, it is desirable to provide a significant increase in overall dynamic range and reduction in channel cross-talk while maintaining sensitivity and compactness of single-spot scanning.